Faster new cloning technique - transcript-based cloning

A single strand of plant or animal DNA may contain tens of thousands of genes, each programmed to produce a specific protein essential for the growth or survival of the organism. The challenge for geneticists is to isolate individual genes and determine their function – a painstaking process often requiring years of laboratory trial and error.

Now an international research team has discovered a technique that dramatically streamlines this process for certain kinds of genes. Developed by scientists at Stanford University and Britain's John Innes Centre, the new procedure could enable scientists to identify specific genes in a matter of months, not years. The technique, known as transcript-based cloning, is described in the March 30 edition of the Proceedings of the National Academy of Sciences (PNAS).

"We believe that this method represents a significant breakthrough in gene cloning," wrote the authors of the PNAS study.

"The greatest impact of this technology is likely to be on plants with large and complex genomes, including most crop species," added Sharon R. Long, the William C. Steere, Jr.–Pfizer Inc. Professor in Biological Sciences at Stanford and co-author of the study. Long, who also serves as dean of Stanford's School of Humanities and Sciences, is an authority on bacterial and plant molecular biology. She and her colleagues used the new cloning technique to isolate and identify a gene in the DNA of Medicago truncatula, or barrel medic – a member of the legume family that is closely related to alfalfa, beans and peas.

"Over the course of six months, we completed what took another group several years to complete, and we identified a pretty cool gene to boot," said Stanford postdoctoral fellow Raka M. Mitra, lead author of the PNAS study. "We think this technology will be applicable to other species and hope that it increases the pace of biological research on the whole."

Reverse genetics

Medicago DNA contains thousands of genes, and using traditional methods to figure out what each one does is a time-consuming process. "The standard approach used by plant geneticists – known as gene cloning – involves breaking down the system in a very controlled way, and then hunting down what's broken," Mitra explained.

This process begins by randomly zapping thousands of plant seeds with radiation, then growing the exposed seeds in a lab. The goal is to raise a mutant plant with an obvious physical mutation, then search through the plant's DNA until the mutated gene of interest is identified. For example, if researchers wanted to find the genes responsible for normal root growth, they would look for a mutant plant with defective roots and then conduct an exhaustive analysis of the plant's DNA until they located the defective genes that caused the damage.

"Gene cloning in M. truncatula can take three to five years, in part because it requires the cross-fertilization of two generations of plants," Mitra said. "I wondered if we could circumvent this laborious hunt for genes. I started from the premise that mutated genes produce mutated proteins – and may even prevent the production of the protein entirely."

In healthy plant and animal cells, protein production begins with the gene – a short stretch of DNA made up of chemicals arranged in a specific sequence that contains the instructions for building the protein. Those instructions are copied from the DNA onto a molecule of RNA in a process called transcription. The RNA copy, or transcript, then moves to another part of the cell, where it is used as a template to manufacture the protein.

Mutated genes, however, carry faulty instructions that produce defective copies of RNA, which the cell tries to eliminate as quickly as possible – a fact that led Mitra and her colleagues to predict that defective RNA would only show up in very low concentrations in mutated cells.

But would the reverse also be true? If a cell produces low quantities of an RNA transcript, does that mean the RNA is the defective product of a damaged gene? If so, researchers could streamline the entire gene-identification process by using reverse genetics. First, they would look for RNAs that occur in low concentrations and determine the chemical sequence of those RNA molecules, then use that information to locate the matching gene on the plant's DNA.

Nitrogen fixation

In the PNAS experiment, Mitra and her coworkers used their new transcript-based cloning technique to identify a plant gene that plays an important role in the production of usable nitrogen for plants and animals. All living things need nitrogen to make proteins. Unfortunately, nitrogen gas, which makes up nearly 80 percent of the atmosphere, is unusable by plants and animals.

However, there are soil-dwelling bacteria that transform atmospheric nitrogen into a compound that plants can absorb in their roots and then convert into proteins – a process called nitrogen fixation. Animals, in turn, get their primary source of nitrogen from plants, which makes bacterial nitrogen fixation essential to all animal – and human – life.

"Our lab has a particular goal – to identify the genes that allow plants to establish beneficial symbiosis for nitrogen fixation, which is also a key to sustainable agriculture," Long noted. As part of that effort, she and her colleagues have been studying the complex chemical signaling that occurs between nitrogen-fixing bacteria and plants. Researchers have discovered that Medicago and other legumes allow bacteria to invade their roots and take up residence in tumor-like organs called nodules.

"This relationship is mutually beneficial," Mitra explained. "The bacteria benefit because they are enclosed in a protective environment – the nodule – where they're fed sugars from the plant. The plant benefits because the bacteria convert nitrogen from the air into ammonia, which the plant uses to make proteins."

Exactly how legumes and bacteria communicate remains something of a mystery. "For unknown reasons, within minutes of recognizing the bacterial chemical signal, calcium levels in the root cells of the plant start oscillating," Mitra said. "These levels rise rapidly, then slowly drop down. This process – called calcium spiking – repeats over and over, at a rate of about one oscillation per minute, and continues for hours."

In an effort to better understand this phenomenon, the research team compared normal Medicago plants with a mutant version raised in the lab. "This mutant was particularly interesting to us because it exhibited calcium spiking behavior but was unable to establish a symbiotic relationship with nitrogen-fixing bacteria," Mitra said.

Using microarray gene-chip technology, the researchers monitored RNA levels produced by 10,000 genes in both normal and mutant plants. "In the mutant plants, we found one gene, called DMI3, that produced extremely low levels of RNA," Mitra said. "The normal version of the DMI3 gene produces a protein that is remarkably similar to tobacco plant proteins that are known to modulate their behaviors in response to calcium."

This finding led the research team to conclude that the DMI3 gene may play an important role in the plant's response to calcium oscillations. Last month, a Dutch and French research team published similar results in the journal Science. However, that group used traditional gene cloning methods to identify DMI3 – a process that took at least four years to complete, compared to six months using transcript-based cloning.

"The bottom line is this," Long said. "In the process of working on nitrogen fixation, we have discovered a general method for identifying and cloning important plant genes that is fast and may be applicable to almost any plant species."